Abstract

A major goal of cancer research is to identify tumor-specific vulnerabilities by which cancer cells can be selectively killed. The clinical success of targeted therapies along with technological advances in genome-wide profiling have spurred large-scale, on-going efforts to systematically map the molecular landscapes of human cancers. These approaches nominate candidate essential or tumor suppressor genes, but are not able to identify all types of acquired vulnerabilities, such as synthetic lethal relationships between aberrantly regulated pathways. Functional studies are therefore an essential complement to these approaches, serving both to confirm the biological roles of candidate cancer genes, and also to identify other vulnerabilities that are acquired as a consequence of observed genetic alterations.

Loss-of-function studies using pooled short hairpin RNAi screening is proving to be a powerful method by which new cancer targets can be identified. In order to identify new vulnerabilities in lung cancer, we have chosen a focused approach using mini-libraries of short hairpins against selected genes of interest. Nuclear hormone receptors and their co-regulators are aberrantly regulated in many cancers, and therapies targeting these receptors are currently used in treatment of breast and prostate cancers. Quantitative PCR expression data across a panel of lung cancer cell lines suggests that nuclear hormone receptors may be useful as prognostic biomarkers in lung cancer patients, but the role of these receptors in lung tumor biology is not fully understood.

We have used a lentiviral pooled short hairpin library of 1062 shRNAs against 120 nuclear hormone receptor and co-regulator genes to identify lung cancer-specific vulnerabilities, both in vitro and in vivo. Briefly, cells were transduced with the library, allowed to undergo a period of selection and expansion, and then injected subcutaneously into NOD-SCID mice. Tumors were harvested after reaching a volume of 300 mm3. A parallel in vitro screen was also performed, in which transduced cells were cultured for 20 population doublings. The relative abundance of individual shRNAs was quantified by next generation sequencing, and genes for which 3 or more shRNAs exhibited depletion either in vitro or in vivo were nominated as candidate essential genes. A screen performed in the lung adenocarcinoma cell line H1819 identified 10 genes that were required for in vitro survival, including BRCA1 and PHB. Five genes were also identified as being required in vivo but not in vitro, including HDAC1 and NCOR2. None of these were found to be mutated by exome sequencing of H1819. Validation studies and investigations into the role for each of these genes in lung cancer are currently on-going. We conclude that our shRNA mini-library screen identifies unsuspected synthetic lethal relationships and thus new therapeutic targets in lung cancer, including some that are only detected by in vivo screening.